diagnostic techniques for inflammatory eye disease past present and future a review

R E V I E W Open AccessDiagnostic techniques for inflammatory eye disease: past, present and future: a review Stephen C Teoh1,2*†and Andrew D Dick3,4† Abstract Investigations used to aid

R E V I E W Open Access

Diagnostic techniques for inflammatory eye

disease: past, present and future: a review

Stephen C Teoh1,2*†and Andrew D Dick3,4†

Abstract

Investigations used to aid diagnosis and prognosticate outcomes in ocular inflammatory disorders are based on techniques that have evolved over the last two centuries have dramatically evolved with the advances in molecular biological and imaging technology Our improved understanding of basic biological processes of infective drives of innate immunity bridging the engagement of adaptive immunity have formed techniques to tailor and develop assays, and deliver targeted treatment options Diagnostic techniques are paramount to distinguish infective from non-infective intraocular inflammatory disease, particularly in atypical cases The advances have enabled our ability

to multiplex assay small amount of specimen quantities of intraocular samples including aqueous, vitreous or small tissue samples Nevertheless to achieve diagnosis, techniques often require a range of assays from traditional

hypersensitivity reactions and microbe specific immunoglobulin analysis to modern molecular techniques and cytokine analysis Such approaches capitalise on the advantages of each technique, thereby improving the

sensitivity and specificity of diagnoses This review article highlights the development of laboratory diagnostic techniques for intraocular inflammatory disorders now readily available to assist in accurate identification of

infective agents and appropriation of appropriate therapies as well as formulating patient stratification alongside clinical diagnoses into disease groups for clinical trials

Keywords: Diagnosis, Uveitis, Ocular inflammation, Hypersensitivity, Polymerase chain reaction, Immunoglobulin, Cytokines, Autoimmunity, Autoregulation

Review

Introduction

Intraocular inflammatory eye diseases though relatively

uncommon remain an important cause of visual

impair-ment For example, uveitis is the third leading cause of

blindness [1-3] Broadly, the underlying aetiologies are

divided into infective and non-infective (presumed

auto-immune or autoinflammatory) causes Since the late 20th

century, advances in molecular techniques have led not

only to increasing our understanding of the pathogenetic

mechanisms that are associated with various forms

non-infectious uveitides, but also to improved refined,

sen-sitive and specific diagnosis of infectious causes Our

understanding of the cellular and molecular pathways

enabled in uveitis has led to the adoption of various immunosuppressive agents to overcome the burden of corticosteroid use, traditional and entrenched in uveitis practice In a recent survey of treatment patterns of non-infectious uveitis by Ophthalmologists in the USA, it was found that up to 60% of patients were still treated with greater than 30mg of steroids for more than 1.5 years as maintenance therapy to control inflammation and the use

of immunosuppressive therapy was only used in 12% of patients 75% of physicians were not aware of treatment guidelines for uveitis [4] Such guidelines are based on data and evidence that include, over time, the iterative bench-to-bedside translation and delivering clinical evi-dence for use of anti-metabolites [5-12] and calcineurin inhibitors [13-16] More recently, progress in targeted therapy with biologics targeted against cytokines (e.g anti-IL-1, anti-IL-6 and anti-TNF-α) [17-24], soluble mediators (e.g interferons) [25,26], or cell surface molecules (e.g Alemtuzumab and CTLA-4 Ig) [27] are showing great promise in the control of refractory non-infective

* Correspondence: Stephen_Teoh@ttsh.com.sg

†Equal contributors

1 National Healthcare Group Eye Institute, Tan Tock Seng Hospital, 11 Jalan

Tan Tock Seng, Singapore 308433, Singapore

2 Eagle Eye Centre, Mount Alvernia Hospital, 820 Thomson Road, Medical

Centre Block B, #02-11/17, Singapore 574623, Singapore

Full list of author information is available at the end of the article

© 2013 Teoh and Dick; licensee BioMed Central Ltd This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and

uveitides There remains the need to provide randomized

controlled trial evidence to confirm their efficacy, some of

which are on going There are increasingly guidelines and

algorithms being developed for immunosuppressive and

immunomodulatory therapies for non-infectious uveitis by

harnessing the increasing evidence being developed, in for

example Behcet’s disease, and adoption by governments

[28] Arguably on the contrary, infective uveitides are still

managed based on the clinician’s experience as such a

clinical diagnosis is sometimes based on clinical signs and

symptoms, supported by demographic information,

mor-phology, laterality and clinical history One clear example

is cytomegalovirus retinitis in HIV [29] However in

prac-tice with many cases, investigations are often necessary to

elucidate and differentiate an aetiology and importantly to

discriminate those that directly cause an infectious disease

versus those evoking an inflammatory disease, such as

la-tent tuberculosis (TB) [30]

In practice, determination of an underlying aetiology is

a routine and important step in the assessment and

evaluation of a uveitic patient 40-86% of patients have

an underlying cause ranging from infectious to

auto-immune causes, whilst the rest remains classified as

idio-pathic when no apparent cause can be identified, but the

condition responds to standard anti-inflammatory

ther-apy [31] Whilst anti-infective agents do not alter the

course or outcome of autoimmune or non-infective

uve-itis, such therapy has no deleterious effects per se on the

condition except that of prolonged and untreated

non-infectious inflammation Conversely, the use of

anti-inflammatory and immunosuppressive agents in infective

uveitides is potentially devastating As such, differentiation

is crucial and defining infectious versus non-infectious

causes is vital from the outset Given the advances in

mo-lecular and cellular pathology and diagnostic ability

ran-ging from laboratory to radiological tests (including X-rays,

computed tomography (CT) scans, magnetic resonance

imaging (MRI) scans, positron emission tomography

(PET) scans and nuclear imaging), we are more enabled to

make such diagnoses In this review, we will focus on the

laboratory, blood and immunological tests, and these will

be further discussed

Infective uveitides vary in prevalence according to

geo-graphic regions Uveitides that were previously

‘undiag-nosed’, labeled and treated as ‘idiopathic’ are increasingly

recognized as related to, or directly caused by an

infect-ive cause as a result of progress in diagnostic techniques

For example, cytomegalovirus detection in aqueous with

resultant therapeutic responses to antiviral agents have led

to improved therapeutic outcomes in hypertensive uveitic

entities such as related syndromes for example,

Posner-Schlossman syndrome [32-34] Fuchs’ heterochromic

iridocyclitis has also been linked to some herpes viruses

and Rubella [35-38], and Tuberculosis-related intraocular

inflammation has seen resurgence in diagnosis following the development of newer diagnostic techniques

Role of diagnostic tests in intraocular inflammation Diagnostic tests in search for an aetiology in intraocular inflammatory diseases have always been controversial, mainly due to its history of suspected lack of specificity and sensitivity of assays Such views have therefore led

to the concept that the need for detecting infectious agents or underlying inflammatory disease, whether for clinical or research purposes, to deliver improved and more tailored diagnosis or understanding of mechanisms

of inflammatory disease must be balanced against the cost of the investigations, the available resources of the treating centre, the utility of the tests employed (given potential lack of sensitivity of assays) and finally, and particularly so in acute circumstances, the time taken to obtain results This is in contrast to performing tests for the overall systemic health of the individual prior to commencement of immunosuppression that can further compromise health In a wider perspective, traditionally

a“textbook” list of relatively untailored investigations re-mains costly and may not until recently, contribute to either diagnosis or change in management A retrospect-ive review of patients with various types of uveitis showed that abnormal values of complete blood counts, plasma viscosity / erythrocyte sedimentation rate (ESR) and VDRL / TPHA did not contribute to establishing an underlying cause of the uveitis [39] A Canadian survey demonstrated that most routine tests performed for the investigation of anterior uveitis lack sensitivity and spe-cificity and have low diagnostic yields [40] In general, investigations are uncommonly performed for anterior uveitides alone except in specific circumstances e.g chronic or recurrent disease, unresponsive or worsening with anti-inflammatory treatment or in hypertensive an-terior uveitides On the other hand, patients with inter-mediate and posterior uveitides, or those patients that present with systemic symptoms and manifestations are usually investigated with a panel of screening tests that comprise an autoimmune and infective screen that typic-ally include syphilis and tuberculosis- two infections that have protean as well as overlapping ocular manifestations Further investigations with blood tests, imaging, molecu-lar diagnosis of aqueous or vitreous samples, or biopsy de-pend on the clinical presentation of the disease

Innate & adaptive immunity in infection Infective pathogens incite inflammatory responses that form the basis of many diagnostic tests The bodies’ nat-ural non-specific antigen-independent innate immunity comprising leukocytes, macrophages and complement activation, interacts with the phylogenetically newer and antigen-specific adaptive immune system comprising

T-and B-cells responses through complex interaction

volving chemokines, cytokines and specialized cells

in-cluding dendritic cells, NK cells and macrophages, in

response to the infectious challenges The measurement

of these responses, both quantitatively and qualitatively,

allows an assessment of the immune status of the

indi-vidual The characteristic granulomatous inflammatory

response generated by the interaction of pathogens and

the CD4+ Th1 cells via IFN-γ following antigen

presen-tation has formed the basis of hypersensitivity tests such

as the Mantoux test Immunoglobulins generated by

ac-tivated B-lymphocytes are routinely detected or

mea-sured that indicate temporal activity of an infection

Advances in technology have also enabled direct

mea-surements of the different levels of cytokines and

chemokines, the relative profiles and levels of which can

be used as adjuncts in the diagnosis of various infections

and inflammatory processes The complex interactions

between innate and adaptive immunity that is hitherto

not fully illuminated, are kept in constant regulatory

checks and balances by a system of chemical mediators

to ensure efficient elimination of pathogens [41-43] A

dysfunctional innate and adaptive immune system on the

other hand, can result in unregulated, inappropriate and

detrimental immune inflammatory responses including

autoimmunity, allergy, allograft rejection and shock [30]

Improvements in diagnostic techniques

Introduction

Diagnostic techniques have evolved from direct

observa-tion of hypersensitivity reacobserva-tions and analyses of

immu-noglobulins, to polymerase chain reactions and the

modern measurements of cytokines Despite the

multi-tude of new tests and techniques, none of the tests are

diagnostic and all are limited by its specificities and

sensi-tivities, and should be interpreted in tandem with clinical

assessment As such, clinicians often use combination

tests, harnessing the different strengths of the tests, to

bet-ter improve the specificity and sensitivity of diagnosis in a

rapid and accurate manner This often involves a mix of

traditional and newer assays

Combination of traditional hypersensitivity tests and

modern cytokine assays

Hypersensitivity responses, a technique that been in use

for the last century, remain commonly used in

combin-ation with modern molecular techniques to assist in the

diagnosis of ocular tuberculosis (TB) TB-related

intra-ocular inflammation is well-known to present in a

myr-iad of protean manifestations Diagnosis has always been

difficult as direct isolation and culture is usually

unavail-able [44] The small tissue and fluid samples that can be

feasibly obtained from ocular samples further limits the

ability to detect the fastidious mycobacterium organisms

Moreover, TB-associated intraocular inflammation is also thought to be immune-mediated, due to reaction to mycobaterial proteins in latent tuberculosis, rather than direct infection [44-46] This often poses a treatment di-lemma between Ophthalmologists and Internists wherein treatment with anti-tuberculosis drugs in these patients with non culture/smear-proven patients are often dis-couraged The classic cornerstone diagnostic test is the tu-berculin skin test (TST) (Mantoux skin test) where tuberculin injected intradermally to produce a localized granulomatous inflammatory response through the inter-action of macrophages and memory Th1 CD4 T-helper lymphocytes in a type IV hypersensitivity reaction The most important limitation of TST is its inability to differ-entiate M tuberculosis and non-tuberculous mycobacterial infections Recent molecular technique advancements in-cluding polymerase chain reaction (PCR) and the use of cytokine analysis in the form of interferon gamma release assays (IGRAs) have been added to the armamentarium of diagnostic tests to increase the specificity and sensitivity of the diagnosis of TB-associated uveitis IGRAs detect the ability of Mycobacterium tuberculosis antigens [early secretory antigen target 6 (ESAT-6) and culture filtrate protein 10 (CFP-10)] to stimulate host production of

IFN-γ, and are superior to TST in distinguishing latent TB in-fections (LTBI) from non-tuberculous mycobacteria and BCG vaccination [47] as it points to exposure to specific tuberculous antigens [48] These antigens distinguish M tuberculosis from most other mycobacteria Although IGRA has not yet been widely tested in subjects with non-tuberculous mycobacterial infection, M kansasii, M szulgai, M marinum, and M bovis may also yield positive results, as they share some common antigens [49,50] However these assays cannot distinguish latent from ac-tive TB infection as positivity merely indicates an exposure

to Mycobacterium tuberculosis Likewise, a positive TST may not distinguish between active disease and atypical mycobacterial infection and a negative avian Mantoux test does not exclude the latter diagnoses [51] There are nu-merous causes for false-positive and false-negative inter-pretations of the TST [52] Even in patients with proven non-tuberculous mycobacterial lymphadenitis, standard TST is only positive in about 50% of cases [53] Each assay, therefore, is limited by its own specificities and sen-sitivities A meta-analysis by Diel et al inferred that IGRAs are superior to TST in diagnosis of active TB [54] Ang et al however reported that TST was more sensitive than T-SPOT.TB (Oxford Immunotec Ltd, Abingdon, UK) but T-SPOT.TB was more specific for diagnosing TB-associated uveitis However a combination of tech-niques involving TST and IGRA is 2.16 times more likely

to diagnose TAU [55] A combination of both TST and IGRA may be useful in distinguishing between tuberculous and non-tuberculous disease, as well as active and latent

disesase In 2007, Gupta synthesized the strengths of these

methods and proposed that a diagnosis of‘presumed’

ocu-lar TB can be made with a consistent clinical presentation

of a granulomatous ocular inflammation alongside a

posi-tive TST or IGRA and/or isolation of mycobacterial DNA

from ocular fluids or tissue using PCR [44,56]

Combination of traditional immunoglobulin analysis and

modern polymerase chain reactions

Immunoglobulin analysis and polymerase chain

reac-tions (PCR) are also commonly combined in the study of

intraocular infection Serological assessment (viz IgG /

IgM) is especially useful in diseases that are not prevalent

or less common in the specific population and

demo-graphics of the patient Coupled with signs consistent and

compatible with an infection, a positive plasma serology

can be interpreted as evidence of an infectious agent in

in-traocular inflammation The observation of

pathogen-specific immunoglobulin isotype class switching from IgM

to IgG in serum, modulated by cytokines including IFN-γ,

IL-4, IL-5 and TGF-β, has been interpreted to be a sign of

recent infection A positive IgM generally indicates

pri-mary or recurrent infection, but may be negative in

im-munocompromised individuals Whereas a positive IgG

suggests seroconversion usually after 2–4 weeks in paired

sera samples or, in the absence of IgM antibodies, is

usu-ally indicative of past infection [57] Within the eye

how-ever, only IgG-class antibody production has been

detected [58] The observation that the amount of this

pathogen-specific intraocular antibody was correlated with

the degree of plasma infiltration within uveal tissue led to

a further refinement with the Goldmann-Witmer

coeffi-cient (GWC) since the 1970s [59-62] PCR, with its high

specificity and ability to analyze small aliquots of samples,

has also been used widely in the aetiological detection of

infective pathogens, masquerade syndromes and

malig-nancies from ocular fluids However, small volumes of

samples are an inherent limitation that can result in

sys-tematic errors and false negatives On the other hand, its

high sensitivity rates can result in false-positive results To

overcome these shortcomings, a combination of GWC

with PCR has been proposed to increase the sensitivity

and specificity of detection [63] De Groot Mijnes reported

a higher detection rate for herpes viruses and toxoplasma

with GWC and PCR assessment [59], and Talabani et al

and Villard et al also reported an increased sensitivity of

80-83% for the detection of toxoplasma infection with

GWC or enzyme-linked immunosorbent assay (ELISA)

and PCR assessment compared to 70-73% with either

technique alone [64,65]

In active endogenous uveitis, elevated immunoglobulins

have also been detected both from the sera as well as

aqueous Elevated IgG, IgM and IgA levels have been

mea-sured in acute anterior uveitis [66-68] Likewise, elevated

non-specific IgG and IgA from aqueous has also been detected It has been proposed that the presence of IgA re-sponse suggests an environmental or infectious aetiology acting across a mucosal tissue However the lack of specificity do not support an infective pathogen-esis On the other hand, the presence of elevated IgG anti-bodies, especially the detection of anti-retinal IgG antibodies, reinforces an autoimmune pathogenesis in

“idiopathic” and non-infectious posterior uveitides Identification of new pathogens with new and combination techniques

The use of modern PCR and traditional immunoglobulin analysis with GWC has also enabled the identification of pathogens in uveitic entities previously thought to be idio-pathic Using GWC techniques, Fuchs’ heterochromic iridocyclitis has been attributed to Rubella and herpes vi-ruses [35-38] With the PCR technique, numerous organ-isms have also been identified from ocular fluids and implicated in ocular inflammation including HTLV-1, ru-bella, Epstein-Barr virus, HHV-6, human parechovirus, dengue and chikungunya virus [69-71] The development

of advanced techniques such as dot hydridization and multiplex PCR has also improved the sensitivity and rate

of detection of several organisms simultaneously while maintaining good sensitivity and specificity [72,73] A sub-set of Posner-Schlossman syndrome (PSS) was found to

be associated herpetic viruses especially cytomegalovirus (CMV) CMV anterior uveitis has since been recognized

as a separate entity with a different clinical course and poorer prognosis compared to PSS, often with more re-lapses and requiring anti-viral therapy [32-34,74] The use

of GWC and PCR has thus improved our understanding

of aetiology and has new bearings on our management of ocular inflammatory diseases

HIV is an increasing worldwide epidemic and is still

on the increase every year [75] The profound systemic immunosuppression in AIDS and immune reconstitu-tion following modern day anti-retroviral therapy (ART) has resulted in a plethora of ocular inflammatory mani-festation ranging from infection to non-infectious im-munogenic immune recovery uveitis [76-81] Not infrequently, ophthalmic manifestations can be the first indicator of HIV disease in patients who have not been previously tested Whilst routine HIV testing is unneces-sary in the assessment of patients with uveitis, a high index of suspicion should be borne in mind in the workup of these patients as there are major implications

on the subsequent management including morbidity and mortality risks in patients who are HIV-positive Patients who should be tested include: 1) patients with known HIV risk factors and high-exposure risk, 2) severe or bi-lateral posterior uveitis, retinitis or choroiditis, 3) fea-tures consistent with CMV retinitis without other

known underlying causes of immunocompromise or

im-munosuppression, [82] 4) concomitant sexually

trans-mitted diseases e.g syphilis, 5) tuberculosis, 6) suspected

herpes zoster uveitis in a young patient < 50 years, and

7) history of constitutional symptoms and unexplained

lymphadenopathy [83]

Role of combination techniques in masquerade syndromes

The use of combination tests to improve the ability to

detect and diagnose is also widely used in masquerade

syndromes and intraocular lymphoma, conditions that

are notorious for difficult diagnosis Current diagnostic

tests include the use of cytopathological analysis [84,85],

flow cytometry [86], PCR demonstrating monoclonality

and IgH gene rearrangements [87], and cytokine analysis

The relative levels of IL-10 vs IL-6 have been utilized as

an adjunct in the diagnosis of primary intraocular

lymph-oma IL-10 is preferentially expressed by B-cell

malignan-cies and acts on B-lymphocytes to stimulate antibody

production In contrast, IL-6 is a principle mediator in

en-dogenous and infective uveitides A ratio of IL-10 to IL-6

levels of greater than 1.0 in both diluted and undiluted

vit-reous samples may act as a diagnostic tool to confirm

in-traocular lymphoma [88-93] Kimura et al found a

detection rate of 91.7% in patients with B-cell lymphoma

with or without vitritis [94] Ohta et al also reported a

sta-tistically significant IL 10:IL 6 ratio in patients with

pri-mary intraocular lymphoma compared to patients with

uveitis (p < 0.0001) [95] However the use of cytokine

ana-lysis alone is controversial as there are still no definitive

diagnostic standards for the use of cytokines in diagnoses

The preparation of vitreous samples for cytopathological

analysis has also changed over time to improve yields from

these limited specimens Intzedy et al reported that

sam-ples placed in saline or prepared fresh followed by paraffin

embedding was able to yield positive diagnosis in all

speci-mens and this has remained the ‘gold-standard’ in

cyto-logical assessment [96] Coupland et al subsequently

proposed that samples for prolonged transport be fixated

with HOPE solution (Herpes-glutamic acid buffer mediated

Organic solvent Protection Effect) improved the quality of

cytomorphology and immunocytology with reduced

arte-facts when compared to unfixed vitreous specimens [97]

Diagnostic tests and techniques have expanded

signifi-cantly, and clinicians are relying on combinations of

tests to increase the sensitivity of detecting an aetiology

Nevertheless all diagnostic tests have their limitations

and should still be interpreted within the clinical context

for consistency [91]

Autoimmunity & autoregulation

The role of autoantigens

The role of autoantigens against various cellular

compo-nents is well-described in connective tissue diseases

including systemic lupus erythematosus (SLE), rheuma-toid arthritis (RA) and Sjogren’s Autoantibodies immu-noglobulins have been commonly used in the supportive diagnosis of connective tissue diseases Rheumatoid fac-tor, an antibody the Fc portion of IgG, is most relevant

in rheumatoid arthritis Other common autoantibodies de-scribed include anti-nuclear antibodies (ANA), double-stranded DNA (dsDNA) in connective tissue diseases and systemic lupus erythematosus, and anti-nuclear cyto-plamsmic antibodies (ANCA) in Wegener’s granuloma-tosis and polyarteritis nodosa, amongst many others These have become common diagnostic tests used by uve-itis specialists when ocular inflammation is the first or only presentation of an auotimmune disease Putative uveitogenic retinal antigens inciting autoreactive lympho-cytes directly or indirectly by antigenic mimicry, such as the soluble Ag (sAg) and interphotoreceptor retinoid-binding protein (IRBP) have also been proposed to be in-volved in idiopathic posterior segment inflammatory conditions although there has been no consistent finding [98-102] More recently dysregulation of the innate im-mune system (autoinflammation) has been recognized to

be the underlying mechanism for various genetic and multifactorial disorders including Blau syndrome and Behcet’s disease resulting in non-specific inflammatory changes due to overexpression of chemokines and cyto-kines including IL-1, IL-6 and TNF-α [30,103] These biomarkers have been described in various intraocular inflammatory conditions and purported to deliver both diagnostic and prognostic uses Li et al suggested that the combination of elevated CXCL10 (>500 ng/mL), CXCL8 (>30 ng/mL) and CCL2 (>60 ng/mL) was a bio-marker to distinguish PSS samples with or without pres-ence of CMV [104] Ang et al found that patients with TB-associated uveitis showed higher levels of IL-6, IL-8, CXCL9 and IP10, and was significantly different from idiopathic uveitis and controls [46] whilst Abu El-Asra found a significant positive association with TAU and IFN-γ, IL-8, MIG and IP-10 suggesting an autoimmune disease rather than an active TB infection Active TB in-fection was typically associated with increased concentra-tions of IL12, TNF-α and IFN-γ [105] Lahmar et al reported that IL-5 and IL-12 were specific for ocular

colony-stimulating factor (GM-CSF) and IL-1 were specific for viral uveitis [106] Jayant et al also demonstrated signifi-cant differences in HIV patients with and without CMV retinitis compared to controls, and this difference con-tinues to persist even in clinically quiescent retinitis [107] Although the use of cytokine and chemokine bio-markers show promise, they still lack true specificity and may represent a pro-inflammatory acute phase reactant The levels of cytokines most likely represent a balance

of type 1 and type 2 cytokines resulting in ocular

inflammation and damage [108] Another potential

diag-nostic use of cytokine biomarkers is in assessment of

clinical resolution of ocular inflammation Current

clin-ical indicator of resolution is based on SUN criteria

[109], but these clinical signs do not predict relapse or

subclinical inflammation Often there are no laboratory

markers of relapse for ocular inflammatory conditions,

and even in AIDS patients on ART, the use of the

‘clas-sical’ CD4 count can fail as a biomarker of immune

re-covery to predict control and suppression of CMV

retinitis infection [110,111] Cytokine and chemokine

markers in these cases may prove to be useful diagnostic

tools As such further work is required to demonstrate

validity of such relatively non-specific biomarkers or

sig-natures for disease types when used either alone or in

combination, for translation into clinical use

Role of genetic factors Genetic and environmental factors are also described in the interaction with autoimmunity Ocular autoimmune disorders have been described to have a MHC class II or

I association, mediating its effects through autoantigens

or cross-reactivity to the MHC motifs from infectious antigens [101] Seronegative arthropathies have been asso-ciated with HLA B27, whilst Birdshot chorioretinpoathy has been associated with HLA A29 [112-114], and a HLA-B*51-restricted peptide from an MHC class-I chain-related gene antigen has been shown to activate CD8+ T-cells with an up-regulated IFN-γ response in Behcet’s disease [115,116] Although PCR analysis for HLA typing has thus been analyzed for pathological associations in ophthalmic disease [117], the use of HLA-typing for diag-nosis is limited and should be interpreted with caution

Table 1 An overview of validity of various tests (and combinations thereof) used in the diagnosis of infective uveitides

Infective agent

Assay n (patients

in studies)

Validity* Reference Ruokuonen et al Rubella (in FHI) Aqueous IgG 63 100.0% [ 35 ]

Aqueous PCR 20 10.0%

Suzuki et al Rubella (in FHI) GWC (> 3) 14 71.4% [ 36 ]

Aqueous PCR 9 22.2%

Quentin et al Rubella (in FHI) AI ( ≥ 1.5) 52 100.0% [ 37 ]

Aqueous PCR 28 17.9%

Ang et al TB IGRA (T-SPOT.TB) 162 Sp 75.0%, Sen 36.0% [ 48 ]

TST Sp 51.1%, Sen 72.0%

TST + T-SPOT.TB OR 2.16 (95% CI, 1.23-3.80)

De Groot-Mijnes et al HSV PCR / GWC + 13 46.2% [ 50 ]

VZV PCR / GWC + 16 62.5%

Toxoplasma PCR / GWC + 25 28.0%

Kiljstra et al / Rothova et al Toxoplasma GWC 22-30 72.7%-93.3% [ 53 , 54 ] Talabani et al Toxoplasma PCR + immunoblotting 54 Sen 73% [ 55 ]

GWC + immunoblotting Sen 70%

PCR + GWC Sen 80%

PCR + GWC + immunoblotting Sen 85%

Villard et al Toxoplasma ELISA 19 Sp 85% [ 56 ]

Immunoblotting 19 Sp 85%

PCR 18 Sp 100%

Dabil et al CMV, VZV, HSV,

Toxoplasma

Multiplex PCR 21 85.7% [ 61 ] Multiplex PCR 71.4% (loss of <1 log sensitivity)

*Most studies are cohort studies and do not represent robust outcomes of validation.

Values stated are positive rates of detection unless otherwise specified.

FHI Fuchs’ heterochromic iridocyclitis, GWC Goldmann-Witmer Coefficient, PCR polymerase chain reaction, AI antibody index, TB Tuberculosis, IGRA interferon-gamma

except B27 in recurrent anterior uveitis in undiagnosed or

misdiagnosed spondyloarthropathies [118] However, in

complex intraocular inflammatory diseases that pose a

diagnostic dilemma, a suggestive HLA typing may be of

value in realigning our differentials The role of HLA B27

may also have limited use in prognostication of anterior

uveitis Accoriniti et al reported a higher incidence of

sys-temic disease (p < 0.001) and 20% required

immunosup-pressive therapy [119] Park et al also reported a higher

incidence of severe anterior chamber activity (p = 0.006),

hypopyon (p = 0.034) and a higher frequency of recurrence

(p = 0.007) [120]

Conclusions

Diagnostic techniques in intraocular inflammation are

constantly developing both technologically and through

our advancements in our understanding of

immuno-logical processes involved What has followed is

develop-ment of such assays that are increasingly specific and

sensitive to various pathologies (Table 1) However,

des-pite the advancements, the clinical practice has to accept

inherent limitations, and manoeuvre between the

false-positives and false-negatives of each test and interpret

results within clinical context Nevertheless, with this

armamentarium of assays and an appropriate utilisation

of combination of these techniques, the uveitis specialist

can move toward more accurate and early

infectious-aetiological diagnosis and toward improving prognosis of

intraocular inflammation as well as increased

categorisa-tion and stratificacategorisa-tion of patients to enable more focused

clinical trials

Competing interests

The authors declare that they have no conflict of interest.

Authors ’ contributions

Both ADD and SCT contributed equally to the concept, preparation and

editing of the manuscript Both authors read and approved the final

manuscript.

Financial disclosure

The authors have no proprietary or commercial interest in any of the

materials mentioned in this article.

Author details

1 National Healthcare Group Eye Institute, Tan Tock Seng Hospital, 11 Jalan

Tan Tock Seng, Singapore 308433, Singapore 2 Eagle Eye Centre, Mount

Alvernia Hospital, 820 Thomson Road, Medical Centre Block B, #02-11/17,

Singapore 574623, Singapore 3 School of Clinical Sciences, University of

Bristol, Bristol Eye Hospital, Lower Maudlin Street, BS1 2LX Bristol, UK.

4 National Institute of Health Research Biomedical Research Centre, Moorfields

Eye Hospital NHS Foundation trust and UCL Institute of Ophthalmology,

London, UK.

Received: 14 January 2013 Accepted: 1 August 2013

Published: 8 August 2013

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doi:10.1186/1471-2415-13-41

Cite this article as: Teoh and Dick: Diagnostic techniques for

inflammatory eye disease: past, present and future: a review BMC

Ophthalmology 2013 13:41.

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